J. AMER . SOC . HORT . SCI . 120(3):481-490. 1995.
Regulation of Fermentative Metabolism in Avocado
Fruit under Oxygen and Carbon Dioxide Stresses
Dangyang Ke, Elhadi Yahia 1, Betty Hess, Lili Zhou, and Adel A. Kader2
Department of Pomology, University of California, Davis, CA 95616
Additional index words. Persea americana, pyruvate decarboxylase, alcohol dehydrogenase, lactate dehydrogenase, pyruvate
dehydrogenase, anaerobic products, pH, NADH, ATP
Abstract. ‘Hass’ avocado (Persea americana Mill.) fruit were kept in air, 0.25% O2 (balance N2), 20 % O2 + 80% CO2, or
0.25% O2 + 80% CO2 (balance N2) at 20C for up to 3 days to study the regulation of fermentative metabolism. The 0.25%
0 2 and 0.25% 02 + 80% CO2 treatments caused accumulations of acetaldehyde and ethanol and increased NADH
concentration, but decreased NAD level. The 20% O2+ 80% CO2 treatment slightly increased acetaldehyde and ethanol
concentrations without significant effects on NADH and NAD levels. Lactate accumulated in avocadoes kept in 0.25 % 02.
The 80% CO, (added to 0.25% O2) did not increase lactate concentration and negated the 0.25% O2-induced lactate
accumulation. Activities of PDC and LDH were slightly enhanced and a new isozyme of ADH was induced by 0.25% O2,
20% O2 + 80% CO2, or 0.25 % O2+ 80% CO2; these treatments partly reduced the overall activity of the PDH complex.
Fermentative metabolism can be regulated by changes in levels of PDC, ADH, LDH, and PDH enzymes and/or by
metabolic control of the functions of these enzymes through changes in pH, ATP, pyruvate, acetaldehyde, NADH, or NAD.
Chemical names used: alcohol dehydrogenase (ADH), adenosine triphosphate (ATP), lactate dehydrogenase (LDH),
nicotinamide adenine dinucleotide (NAD), reduced NAD (NADH), pyruvate decarboxylase (PDC), pyruvate dehydrogenase (PDH).
Short-term exposure of fruit to very low 02 and/or very high
C O2 concentrations may have potential benefits for insect
disinfestation, disease control, and alleviation of some physiological disorders. However, some undesirable physiological changes
may also occur in plant tissues under stress conditions of O2 and
CO 2. Yahia and Carrillo-López (1993) observed exocarp and
mesocarp injury on ‘Hass’ avocado fruit exposed to 0.1% to 0.4%
O 2 + 50% to 75% CO2 at 20C for longer than one day before
ripening in air.
Plant responses to very low O2 and/or very high CO2 concentrations include induction of fermentation pathways, accumulation of
succinate and/or alanine, and decreases in intracellular pH and
ATP levels. One pathway of fermentative metabolism results in
accumulation of acetaldehyde and ethanol catalyzed by the enzymes PDC and ADH, respectively (Ke et al., 1994). In some plant
tissues, lactate accumulation results from fermentation and this is
catalyzed by the enzyme LDH. The major function of fermentative
metabolism is to use NADH and pyruvate when electron transport
and oxidative phosphorylation are inhibited so that glycolysis can
proceed. This will allow for the production of some ATP through
substrate phosphorylation, which permits the plant tissues to
survive temporarily. Kanellis et al. (1991) found that ADH isozymes
could be induced by exposure of avocado fruit to 2.5%, 3.5%, or
5.5% O2. Increased activities of PDC and ADH were observed
when sweetpotato, ‘Bartlett’ pear, lettuce, and strawbeq were
kept in low O2 or high CO2 concentrations (Chang et al., 1983;
Nanos et al,, 1992; Ke et al., 1993, 1994). In maize, barley, and rice,
the increases in activities of PDC, ADH, and LDH by low O2 have
been found to be due to increased transcription and translation of
the related genes, resulting in new mRNA synthesis and de novo
synthesis of the corresponding enzyme proteins (Gerlach et al.,
Received for publication 19 Sept. 1994. Accepted for publication 10 Jan. 1995.
Research supported in part by U.S. Dept. of Agriculture research agreement no, 58319R-3-004. The cost of publishing this paper was defrayed in part by the payment
of page charges. Under postal regulations, this paper therefore must be hereby
marked advertisement solely to indicate this fact.
1
Current address: Universidad Autonoma de Queretaro, Centro Universitario,
Cerro de Las Campanas, Queretaro 76040, Mexico.
2
To whom reprint requests should be addressed.
J. AM E R. SO C. HORT . SCI . 120(3):481-490. 1995.
1982; Good and Crosby, 1989; Kelley, 1989).
In this study, we investigated the regulation of ethanol and
lactate fermentation in avocado fruit in response to low O2 and/or
high CO2 stresses.
Materials and Methods
Materials and treatments. ‘Hass’ avocado fruit were harvested
at commercial maturity from an orchard near Santa Barbara, Calif.,
and shipped to our laboratory in Davis, Calif., where they were
stored in air at 5C for <1 week before use for experiments. Nine
fruit were placed in a 4-liter glass jar and ventilated with humidified air or a specified gas mixture at a continuous 100 ml·min-1 flow
rate. The gas mixtures included 0.25% O2 (+ 99.75% N2),20% O2
+ 80% CO2, and 0.25% O2 + 80% CO2 (+19.75% N2). Three
replicates were used for each treatment. The fruit samples were
kept in air or the specified atmospheres at 20C for 1,2, or 3 days
followed by transfer to air at 20C for another 3 days to observe the
post low 0 2 and/or high CO 2 effects.
Extraction and measurements of fermentation products. Volatiles were extracted by homogenizing 10 g fruit tissue in 15 ml of
0.1 M HC1 solution. The homogenate was stored at –40C for <1
month before use for analysis of acetaldehyde and ethanol by a gas
chromatography (HP5890A; Hewlett Packard, Palo Alto, Calif.).
To extract lactate, 6 g fruit tissue was added to a 50-ml centrifuge
tube containing 12 ml of 1.8% (w/v) Ba(OH)2. The sample was
homogenized and then 12 ml of 2% (w/v) aqueous ZnSO4 solution
was added and immediately shaken for 15 sec. The mixture was
filtered through four layers of cheesecloth and centrifuged for 15
min at 27,000× g. The supernatant was decanted, filtered through
four layers of cheesecloth, and used for analysis of lactate by the
enzymatic method of Leshuk and Saltveit ( 199 1). Recovery rate of
lactate was 78% from the extraction procedure.
Extraction and assays of fermentation enzymes. For each replicate, 6 g tissue (six discs) were obtained from three fruit and
homogenized in 20 ml of 100 mM 2-(N-morpholino) ethane-sulfonic acid (MES) buffer (pH 6.5) containing 2 mM dithiothretol and
1% (w/v) PVP. The homogenate was filtered through four layers
of cheesecloth and centrifuged at 27,000× g for 10 min. The
481
supernatant was retained as enzyme extract for the measurements
of ADH, LDH, and PDC activities. ADH activity was measured by
mixing 0.88 ml of 100 m MMES buffer(pH 6.5), 0.06 ml of 1.6 mM
NADH, 0.01 ml of enzyme extract, and 0.05 ml of 80 m M
acetaldehyde. LDH activity was measured by mixing 0.79 ml of
100 mM MES buffer (pH 6.5), 0.025 ml of 100 mM 4-methylpyrazol,
0.025 ml of 100 mM KCN, 0.05 ml of 1.6 mM NADH, 0.06 ml of
50 mM Na pyruvate, and 0.05 ml of enzyme extract. For ADH and
LDH, oxidation of NADH was measured by recording the decrease in absorbance at 340 nm using a spectrophotometer. PDC
activity was assayed by measuring acetaldehyde production of the
enzyme reaction. The PDC reaction mixture was obtained by
mixing 3.2 ml of 100 mM MES buffer (pH 6.5), 0.5 ml of 5 mM
thiamine pyrophosphate (TPP), 0.5 ml of 50 mM MgCl2, 0.5 ml of
enzyme extract, and 0.3 ml of 50 mM Na pyruvate in a 15-ml screwcap test tube. The sample was incubated at 30C for 60 min and a
1 -ml headspace gas sample was taken for analysis of acetaldehyde
concentration by a gas chromatography. The conventional method
of measuring PDC by coupling PDC and ADH reactions through
using pyruvate and NADH as substrates did not work with avocado
since its high LDH activity competed and interfered with the
coupled PDC and ADH reactions. Enzyme activities of PDC,
ADH, and LDH were expressed as mmoles of product formed or
substrate used per min per gram fresh weight. For in vitro studies
of enzyme kinetics, several assay pH values and concentrations of
pyruvate, acetaldehyde, NADH, NAD, and ATP were used to
study the changes in activities of PDC, ADH, and LDH.
Extraction, electrophresis, and staining of ADH isozymes.
Three grams of fruit tissue (three discs) were homogenized in 12
ml extraction buffer and 1 g PVPP. The homogenate was filtered
through four layers of cheesecloth and centrifuged at 27,000× g for
10 min. The supernatant was retained for isozyme analysis by
starch gel electrophoresis. The gels were prepared, run, and ADH
isozymes stained according to Arulsekar and Parfitt ( 1986).
Isolation and measurements of NAD and NADH. The method of
Greenbaum et al. (1965) was modified. Six grams of fruit tissue ( 12
half discs) were frozen in liquid nitrogen and then ground to
powder. The powder was transferred to a 50-ml centrifuge tube
that contained 12 ml of 0.1 N HCl for extraction of NAD. Similarly,
the other 12 half discs (6 g) were frozen in liquid nitrogen, ground
to powder, and then placed in another 50-ml centrifuge tube that
contained 12 ml of 0.1 N NaOH for extraction of NADH. After 20
min on ice with periodic stirring, the tubes were soaked in boiling
water for 2 min and immediately transferred to ice and kept there
for 15 min. The samples were filtered through four layers of
cheesecloth and then 1 ml of 500 mM potassium phosphate buffer
(pH 7.5) was added to each tube. The samples were neutralized to
pH 7.3–7.5 while stirring on ice by dropwise addition of 1 N NaOH
to the acid extract and 1 N HCl to the alkaline extract, respectively.
The samples were centrifuged at 27,000× g for 10 min at 4C. The
supernatant was decanted, filtered through four layers of cheesecloth, and kept on ice for immediate assays of NAD and NADH.
NAD or NADH concentration of each sample was measured in a
very sensitive cyclic enzyme reaction system where NAD or
NADH was used as a rate limiting component. The reaction
mixture was obtained by mixing 0.63 ml of 100 mM potassium
phosphate buffer (pH 7.5),0.03 ml of 60 mM phenazine methosulfate
(PMS), 0.04 ml of 0.6 mM dichlorophenol indolphenol (DCPIP),
0.1 ml of 95% ethanol, 0.1 ml of commercial ADH solution (5
units), and 0.1 ml of sample extract. In this system, ethanol is
oxidized to acetaldehyde by ADH with the reduction of NAD to
NADH. NADH transfers protons and electrons to PMS and NAD
is recycled for the ADH reaction. The reduced PMS transfers
482
protons and electrons to DCPIP. Reduction of the blue-colored
DCPIP to colorless DCPIPH, was measured by recording the
decrease in absorbance at 600-nm. A standard curve for NAD or
NADH over a range of 0.002–0.015 mM was obtained for calculating NAD or NADH concentration of the sample. Recovery rates of
NAD and NADH from the extraction procedures were 83% and
78%, respectively.
Mitochondria isolation. Mitochondria were isolated from fruit
tissue using the method of Morea and Romani (1982) with slight
modification. Thirty grams of fruit tissue were macerated in 100 ml
isolation medium which consisted of 50 mM potassium phosphate
buffer (pH 7.2), 0.25 M sucrose, 5 mM EDTA, 4.5 mM ß mercaptoethanol, 0.2% PVP (w/v) and 0.1% BSA (w/v). The
homogenate was filtered through four layers of cheesecloth and
centrifuged at 2,000× g for 10 min. The supernatant was filtered
through four layers of cheesecloth and centrifuged at 10,000× g for
5 min. This supernatant was discarded and the pellet was resuspended in 1 ml wash medium and homogenized with a glass
microhomogenizer in the presence of an additional 3 ml wash
medium. The wash medium contained 50 m potassium phosphate buffer (pH 7.2), 0.25 M sucrose, and 0.1%. BSA. The
homogenate was centrifuged at 2000× g for 5 min and the supernatant was recentrifuged at 8000× g for 10 min. The pellet was
resuspended in 0.5 ml wash medium and used as mitochondrial
preparation. Protein content of mitochondrial preparation was
determined by the standard Bradford (1976) method.
Assay of PDH. The overall activity of the PDH complex was
assayed using the method of Budde and Randall (1987) with some
modification. The reaction mixture was obtained by mixing 0.55
ml of 200 mM potassium phosphate buffer (pH 8.0), 0.05 ml of 4%
(v/v) Triton X- 100,0.05 ml of 50 mM MgCl 2, 0.05 ml of 40 mM
NAD, 0.05 ml of 5 mM TPP, 0.05 ml of 2.5 mM LiCoA, 0.05 ml of
20 mM cysteine, 0.05 ml of 20 mM Na pyruvate, and 0.1 ml of
mitochondrial preparation. Formation of NADH was measured by
recording the increase in absorbance at 340 nm using a spectrophotometer. PDH activity was expressed as mmoles of product formed
per min per mg protein.
Results and Discussion
Changes in fermentation products and enzymes. Avocado fruit
exposed to 0.25% O2 or 0.25% O2 + 80% CO2 at 20C for 1 to 3 days
accumulated acetaldehyde (Fig. 1A) and ethanol (Fig. 1 B). The
fruit kept in 0.25%02 also accumulated lactate (Fig. 1C). After the
fruit were transferred from 0.25% O2 or 0.25% O2 + 80% CO2 to
air for 3 days, the accumulated fermentation products largely
disappeared, probably as a result of their metabolism to other
compounds or due to diffusion of acetaldehyde and ethanol to
outside the fruit tissue. The 20% O2+ 80% CO2 treatment did not
significantly increase acetaldehyde and ethanol concentrations
and it did not induce lactate accumulation at all. Also, the 80% CO2
negated the effect of 0.25% O2 on lactate accumulation (Fig. 1C).
After exposure of avocado fruit to 0.25% O2, 20% O2 + 80%
CO2, or 0.25% O 2 + 80% CO2 for 2 to 3 days, extractable activities
of PDC and LDH were slightly higher than those of air control fruit
(Fig. 2 A and C). The low O2 and/or high CO2 treatments did not
significantly influence extractable activity of ADH (Fig. 2B).
However, isozyme analysis indicated that a new ADH isozyme
appeared in the fruit exposed to 0.25% O2, 20% O2 + 80% CO2, or
0.25% 02+ 80% CO2 for 2 days compared to the ADH isozyme
profile of air control fruit (Fig. 3). The ADH activity in avocado
fruit was about 10 times that of PDC activity and about 20 times
that of LDH activity (Fig. 2). Furthermore, ADH activity of
J. AMER . SO C. HO R T. SCI . 120(3):481-490. 1995.
avocado was 40 to 80 times higher than that of ‘Bartlett’ pear (Ke
et al., 1994), lettuce, and strawberry (Ke et al., 1993). The extremely high ADH activity in avocado fruit probably made it
difficult to see any small change in the total enzyme level since the
new ADH isozyme band had a very low intensity (Fig. 3).
Kennedy et al. (1992) reviewed studies of anaerobic metabolism in plants under low O2 stress and concluded that the induction
of PDC, ADH, and/or LDH was one of the mechanisms for
accumulations of anaerobic products. Increases in activities of
these enzymes by low O2 have been found to be largely due to
increased transcription and translation, resulting in new mRNA
synthesis and de novo synthesis of the corresponding enzyme
proteins (Gerlach et al., 1982; Good and Crosby, 1989; Kelley,
1989). Ke et al. (1993) found that 0.25% O2 increased extractable
activity of ADH several fold in ‘Bartlett’ pear, lettuce, and strawberry, largely due to the induction of one ADH isozyme. But 20%
O2 + 80% CO2had only slight effect on ADH activity of pears. The
effects of 0.25% O and/or 80% CO2 on extractable activities of
PDC and LDH of these commodities were also slight. Since
exposure of avocado fruit to 0.25% O2 for 3 days increased the
concentrations of acetaldehyde, ethanol, and lactate over 8-, 172-,
and 33-fold, respectively (Fig. 1), but had only slight effects on the
levels of PDC, ADH and LDH (Figs. 2 and 3), it appeared that
molecular induction of the expression of these fermentation en2
Fig. 1. Changes in concentrations of acetaldehyde, ethanol, and lactate of ‘Hass’ avocado fruit kept in air, 0.25% O2, 20% O2+ 80% CO2, or 0.25% O 2 + 80% CO2 at 20C
for 1, 2, or 3 days followed by transfer (indicated by arrow) to air at 20C for 3 days. The vertical bars represent LSD at P = 0.05.
J. AMER . SOC . HORT . SCI. 120(3):481-490. 1995.
483
zymes was not the major mechanism for regulating fermentative
metabolism in avocado fruit. Roberts et al. (1989) and Xia and
Saglio (1992) also suggested that activities of ADH and LDH were
not the rate limiting factors for accumulations of ethanol and
lactate in some plant tissues if the activities of these enzymes were
high. We tested the possibility that other factors might be more
important for induction of fermentation in avocado fruit.
Changes in fermentation substrates and cofactors. Avocado
fruit kept in 0.25% O2, 20% O2 + 80% CO2, or 0.25% O2 + 80% CO2
at 20C for 3 days had lower PDH activity than that of air control
fruit (Table 1). This suggested that pyruvate flux through the
tricarboxylic acid (TCA) cycle might have been reduced by the low
O2 and/or high CO2 treatments. As a result pyruvate concentration
in the treated fruit could have been increased. Davis et al. (1973)
observed that pyruvate content was higher in low O2 or high CO2treated citrus fruit than that of air control fruit. Recent] y, Good and
Muench ( 1993) reported that levels of pyruvate as well as those of
lactate and ethanol rapidly increased in barley root tissue exposed
to anaerobic conditions.
NADH is the common substrate for ADH and LDH. Exposure
of avocado fruit to 0.25% O2 or 0.25% O2 + 80% CO2 increased
NADH concentration but decreased NAD level (Table 1), which is
correlated with the accumulation of ethanol in these fruit (Fig. 1 B).
The 20% O2 + 80% CO2 treatment had no significant effect on
Fig. 2. Changes in activities of pyrovate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH) of ‘Hass’ avocado fruit kept in air, 0.25%
O2, 20% O2+ 80% CO2, or 0.25% O2 + 80% CO2at 20C for 1,2, or 3 days followed by transfer (indicated by arrow) to air at 20C for 3 days. The vertical bars represent
LSD at P = 0.05.
484
J. AM E R. SO C. HO R T. SCI . 120(3):481-490. 1995
Fig. 3. Isozymes of alcohol dehydrogenase
(ADH) of ‘Hass’ avocado fruit kept in
air (lane A), 0.25% O2 (lane B), 20% 0 2
+ 80% CO2 (lane C) or 0.25% O2 + 80%
CO2 (lane D) at 20C for 2 days, -
NADH and NAD contents, consistent with its slight effect on
ethanol concentration. Similarly,
the NADH : NAD ratio was
greatly increased by the 0.25%
O2 or 0.25% O2 + 80% CO2 treatment but not by the 20% O2 +
80% CO2 treatment (Table 1).
These results indicated that the
decrease in O2 concentration
played a major role in increasing
concentrations of fermentation
substrates and in accumulations
of fermentation products in avocado fruit. This is in contrast to
some other fruit, such as ‘Bartlett’ pear and strawberry, where 80%
CO 2 induced large accumulations of acetaldehyde and ethanol
even under 207002 (Ke et al., 1993).
We have reported that avocado fruit had a cytoplasmic pH of 6.9
and that exposure to 0.25% O2, 20% O2 + 80% CO2, or 0.25% O2
+ 80% CO2 decreased the pH values to 6.7, 6.3, and 6.3, respectively (Hess et al., 1993). Reduced O2 or elevated CO2 concentrations also decreased cytoplasmic pH in ‘Bartlett’ pear (Nanos and
Kader, 1993) and lettuce (Siriphanich and Kader, 1986). These
decreases in cytoplasmic pH may play an important role in regulating fermentative metabolism (Davies, 1980; Roberts, 1989).
Under O2 levels near 0% or high CO2 concentrations, electron
transport through the cytochrome pathway was inhibited (Kennedy
et al., 1987). As a result, oxidative phosphorylation would be
decreased. Nanos and Kader(1993) found that exposure of ‘Bartllet’
pear to 0.25% O2 greatly reduced the ATP/ADP ratio. Exposure of
avocado fruit to 0.25% O2 or 20% O2 + 80% CO2 reduced ATP level
by 20% and 22%, respectively (Hess et al., 1993); the combination
of 0.25% O2 +80% CO2 had a synergistic effect on inhibiting ATP
synthesis, resulting in a 63% reduction in ATP level. Kerbel et al.
(1988) reported that exposure of ‘Bartlett’ pear to 10% CO2
increased fructose-6-phosphate concentration but decreased fructose- 1,6-bisphosphate level, consistent with a reduction in activities of ATP: phosphofructokinase and PPi : phosphofructokinase.
Metabolic regulation of fermentative metabolism. Fermentative metabolism can be regulated by two mechanisms: molecular
control (also called coarse control) of the levels of PDC, ADH, and
LDH and metabolic control (also called fine control) of the actual
functions of these enzymes in plant tissue under O2 and/or CO2
stresses (John and Greenway, 1976; Chang et al., 1983; Ke et al.,
1993). Roberts et al. (1 989) found that ethanol production rate was
correlated with ADH activity when the enzyme level was very low:
but at a high enzyme level, e’thanol accumulation was independent
of ADH activity. With limited enzyme level, the induction of a
fermentation enzyme through molecular control (transcription
and/or translation) is essential for the accumulation of fermentation products. If the enzyme level is already very high even under
air control like the ADH activity in avocado fruit, then it may not
be critical to induce more enzyme biosynthesis. On the other hand,
metabolic control of the actual functions of the fermentation
enzymes by changes in pH and concentrations of substrates,
cofactors, and/or inhibitors may always be important in regulating
fermentative metabolism.
The optimum pH for PDC of avocado fruit was about 6.0 to 6.5
(Fig. 4A). A decrease in cytoplasmic pH from 6.9 to 6.7 by 0.25%
02 or to 6.3 by 80% CO2 (Hess et al., 1993) would activate PDC.
Davies (1980) and Roberts (1989) proposed that a decrease in
cytoplasmic pH and the subsequent activation of PDC was the
major reason to direct pyruvate to the production of acetaldehyde
and ethanol in plant tissue. The optimum pH for ADH and LDH of
avocado fruit was about 6.5–7.0. A decrease in cytoplasmic pH of
0.2 units by 0.25% O2 would not significantly influence LDH
activity but would slightly inhibit ADH (Fig. 4A). A decrease in
cytoplasmic pH from 6.9 to 6.3 by 80% CO2 would significantly
inhibit ADH and LDH of avocado fruit (Fig. 4A), ADH activity
(Fig. 2B) was 10 times higher than PDC activity of avocado fruit
(Fig. 2A). Therefore, a partial inhibition of ADH by a decrease in
pH would not limit the conversion of acetaldehyde into ethanol. On
the other hand, since LDH activity (Fig. 2C) was only about half
that of PDC activity (Fig. 2A) in avocado fruit, the inhibition of
LDH by a decrease in pH from 6.9 to 6.3 due to 80% CO2 might
have a greater impact on LDH, which in turn favored PDC action.
This may partly explain why 0.25% O2 caused accumulations of
acetaldehyde, ethanol, and lactate but the combination of 0.25% O2
+ 80% CO2 resulted in accumulations of acetaldehyde and ethanol
only (Fig. 1). In other words, 80% CO2 decreased cytoplasmic pH
to a level that significantly inhibited LDH and therefore negated
the effect of 0.25% O2 on lactate accumulation. In the range of pH
6.0–8.0, a decrease in pH would inhibit PDH (Fig. 4A) and
therefore reduce the pyruvate flux through the TCA cycle.
LDH was inhibited at high ATP concentrations (Fig. 4B). ADH
and PDH were slightly inhibited by 1 mM ATP but not by the other
ATP concentrations used. High ATP concentrations slightly inhibited PDC. Oba et al. (1977) and Betsche (198 1) also reported that
LDH was inhibited at high ATP level. Exposure of avocado fruit
to 0.25% O2 reduced ATP level (Hess et al., 1993), which would
have relieved the effect of ATP on inhibiting LDH and partly
contributed to the accumulation of lactate in the tissue (Fig. 1C).
It was reported that fructose-6-phosphate relieved the ATP inhibition of LDH in lettuce (Betsche, 1981) and 10% CO2 treatment
caused an increase in concentration of fructose-6-phosphate (Kerbel
et al., 1988).
Table 1. Effects of exposure to air, 0.25% O2, 20% O2+ 80% CO2, or 0.25% O2 + 80% CO2 for 3 days at 20C on overall activity
of pyruvate dehydrogenase (PDH) complex and concentrations of NAD and NADH of avocado fruit.
Treatment
Air
0.25%0,
20% O2 + 80% CO2
0.25% O2 + 80% CO2
LSD at P = 0.05
PDH activity
(µmol·min -1· m g -1)
0.015
0.007
0.007
0.009
0.005
J. AMER . SO C. HORT . SCI . 120(3):481-490. 1995.
NAD
( mM )
0.064
0.020
0.059
0.023
0.009
NADH
( mM )
0.002
0.008
0.003
0.011
0.003
NADH : NAD
ratio
0.03
0.38
0.05
0.50
0.09
485
Fig. 4. Activities of pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and pyruvate dehydrogenase (PDH) in ‘Hass’ avocado
fruit as influenced by pH and ATP concentration. PDH was incubated with specified ATP concentrations for 90 min at 20C before assay. All the other enzymes were
assayed immediately following ATP addition.
Pyruvate is the common substrate for PDC, LDH, and PDH.
The Km of LDH for pyrttvate was 0.08 mM (Table 2). LDH was
more preferred than PDC at lower pyruvate concentration (Fig.
5A). At higher pyruvate level, however, PDC would be more
active than LDH. PDH also had a low Km for pyruvate (Table 2)
and at normal physiological conditions of air control fruit pyruvate
was expected to be largely directed to the PDH reaction and
subsequently to TCA cycle. However, since 0.25% O2 and/or 80%
CO2 treatment reduced PDH activity (Table 1) and a decrease in pH
inhibited PDH (Fig. 4A), pyruvate flux through the PDH path
could have been reduced, which in turn might have increased
pyruvate concentration. This would activate PDC and LDH since
both are allosteric enzymes that could be activated by their substrate (pyruvate) due to confirmational changes (Betsche, 1981;
Hubner et al., 1978).
ADH of avocado fruit had a Km of 1.22 mM for acetaldehyde
(Table 2). In air control fruit, acetaldehyde concentration was
much lower than the Km value (Fig. 1A). The 0.25% O2 or 0.25%
O2+ 80% CO2 treatment increased acetaldehyde concentration to
0.2–0.5 mM in 1 to 3 days, which would have greatly accelerated
the ADH reaction and the accumulation of ethanol (Fig. 5B). Since
ADH activity in avocado fruit was very high (Fig. 2B), ADH was
not likely to be the factor that limited ethanol production. Instead,
the increased acetaldehyde concentration was probably the major
Table 2. Km values of pyruvate decarboxylase (PDC), alcohol dehyrogenase (ADH), lactate dehydrogenase (LDH), and pyruvate
dehydrogenase (PDH) of avocado fruit for their substrates.
486
J. A MER . SOC . HORT . SCI . 120(3):481-490. 1995.
Fig. 5. Activities of pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH) in ‘Hass’ avocado fruit as influenced by pyruvate
or acetaldehyde concentration
driving force for ethanol accumulation in avocado fruit kept in
0.25% O2 or 0.25% O2 + 80% CO2 (Fig. 1 A and B and Fig. 5B).
NADH could be the substrate for ADH or LDH. Without the
addition of NAD, the Km values of ADH and LDH for NADH were
0.019 and 0.030 mM, respectively (Table 2, Fig. 6A). This indicated that ADH of avocado fruit had a higher affinity for NADH
than that of LDH. Addition of 0.08 mM NAD increased the Km
values of ADH and LDH for NADH to 0.034 and 0.091 m M ,
respectively. The inhibition of NADH oxidation by NAD was
more pronounced at lower NADH concentrations, such as 0.01 or
0.02 mM, than at higher NADH concentrations, such as 0.04 or 0.08
mM (Fig. 6 B and D). It was difficult to know the actual NADH and
NAD concentrations in cytoplasm and mitochondria of avocado
fruit since the vacuole usually occupies the major volume of a
mature fruit cell. However, the decrease in NAD concentration in
avocado fruit kept in 0.25% O2 or 0.25% O2 + 80% CO2 (Table 2)
would have relieved the effect of NAD on inhibiting NADH
oxidation by ADH or LDH. The increase in NADH concentration
by 0.25% O2 (Table 1) was probably an important driving force for
the ADH or LDH reaction.
Lactate concentration was 6 times higher than acetaldehyde
concentration in the avocado fruit kept in 0.25% O2 for 2 to 3 days
J. AMER . SOC . HORT . SCI. 120(3):481-490. 1995.
(Fig. 1). However, the total pyruvate flux to the ethanol pathway
was 10 times higher than that to the lactate pathway. Although
LDH had a higher affinity for pyruvate (Table 2), the PDC activity
was twice that of LDH (Fig. 2). A decrease in cytoplasmic pH
substantially activated PDC but not LDH (Fig. 4A). Furthermore,
ADH had a higher affinity for NADH than that of LDH and the
acetaldehyde produced from PDC reaction was easily converted
into ethanol due to the high level of ADH activity. Therefore, the
ethanol pathway was more preferred than the lactate pathway in
avocado fruit as in other plant tissues such as ‘Bartlett’ pear,
lettuce, and strawberry.
From the results of this paper and cited references, a proposed
mode of action of low O2 stress on fermentative metabolism in
avocado fruit is presented in Fig. 7. The low O2 concentration
substantial y reduces NADH flux through the electron transport
system (ETS). As a result, concentrations of NAD and ATP
decrease while NADH level increases. Cytoplasmic pH is decreased, PDH activity is partly reduced and pyruvate flux through
the TCA cycle decreases. PDC and LDH activities are enhanced
and a new ADH isozyme is induced. PDC activity is enhanced by
the decrease in pH and by an increase in pyruvate concentration.
This directs pyruvate to the production of acetaldehyde. Although
487
Fig. 6. Activities of alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) in ‘Hass’ avocado fruit as influenced by concentrations of NADH and NAD
a decrease in pH slightly inhibits the very abundant ADH, the
increased acetaldehyde and NADH concentrations and decreased
NAD level drive ADH reaction, which causes ethanol to accumulate as the major fermentation product. The increase in pyruvate
and NADH concentrations and decreased NAD and ATP levels
activate LDH and drive pyrtrvate conversion into lactate as another
end product. The induction of fermentative metabolism allows
glycolysis to go on, NADH and pyruvate can be used, and a small
amount of ATP can be produced through substrate phosphorylation to permit the plant tissue to temporarily survive. However, the
accumulations of acetaldehyde, ethanol, and lactate and the disturbance of normal metabolism maybe detrimental to the avocado
fruit if it is exposed to low O2 stress conditions beyond its limit.
Although the 20% O2 + 80% CO2 treatment induced or activated
PDC, ADH, and LDH (Figs. 2-4), it did not cause significant
changes in NADH, NAD, and lactate (Table 1, Fig. 1C) and only
slightly increased acetaldehyde and ethanol concentrations (Fig. 1
488
A and B) in avocado fruit. The avocado responses to 20% O2 + 80%
CO2 were different from those of pears and strawberries m which
20% O2+ 80% CO2 induced great accumulations of acetaldehyde
and ethanol (Ke et al., 1993, 1994). This suggested that under 20%
02+ 80% CO2, electron transport in avocado fruit might be only
partially inhibited and/or another pathway could have been induced to replace fermentative metabolism to use pyruvate and
NADH and to produce ATP.
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Fig. 7. Pathways of fermentative metabolism in ‘Hass’ avocado fruit as proposed to be regulated by low O 2 stress. ADH = alcohol dehydrogenase; ETS = electron transport
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